PROJECT SUMMARY/ABSTRACT The objective of this proposal is to move our product forward toward commercialization by improving the quality of life for breast cancer chemotherapy patients. This will be achieved by the following Specific Aims: Aim 1 will optimize differentiation of GMPs and neutrophil lineages in culture in response to G-CSF. This will be assessed by flow cytometry to quantify production of neutrophil lineages and by colony forming assays to quantify lineage multipotency. Aim 2 will identify the optimal extent of lineage differentiation for production of GMPs and neutrophil lineages to prevent neutropenia in the busulfan cancer chemotherapy treatment of NSG mice. Efficacy will be quantified by production of human cells in peripheral blood, bone marrow and spleen using flow cytometry. These studies will define the process to manufacture a frozen, off-the-shelf product to directly transfuse into chemotherapy patients at increased risk of infection. The significance of our proposal is based on the strong scientific premise of extensive literature and our preliminary data. Our finding has the potential to revolutionize the value of CB stem cells in transplant indications by filling the unmet clinical need for more cord blood-derived cells. The need for more cells has long been extolled but ex vivo manipulations have only brought incremental improvements. Our technology provides unprecedented numbers of stem, progenitor and immune cells to patient quality of life at a remarkably reduced cost from scale-up manufacturing. The proposed studies will leverage this next generation expansion discovery to address the problem of reduction of GMPs after chemotherapy, neutropenia, and consequent increases in infection. This, in turn, is a significant problem because infections compromise maintenance of chemotherapy regimens, thus resulting in poorer outcomes. Further, the proposed studies embody a rigorous scientific approach based on state-of-the-art literature. This innovation is truly a paradigm shift because previously the limited numbers of CB cells has limited this valuable, efficacious cellular therapy from impacting larger numbers of patients. This work will complete preclinical, in vivo, proof-of-principle studies in mice. If successful, this will have a major impact on chemotherapy patients by leading to a reduction in A) infections that compromise maintenance of the chemotherapy regimen in these patients, B) hospitalization times, C) cost, and D) morbidity and mortality. Ultimately, the proposed studies will accelerate Cord Blood Plus, Inc. closer to migration to cGMP production, a pre-IND meeting with the FDA, and design of a protocol for a Phase 1 safety and tolerability clinical trial through our new collaboration between MD Anderson Cancer Center and UTMB.